Beta-rhodomycin v

ABSTRACT

An antibiotic designated Beta -rhodomycin V and corresponding to the formula WHEREIN R represents a tetraglycoside consisting of 2 mols rhodinose, 1 mol 2-desoxy-fucose and 1 mol rhodosamine is prepared by cultivating Streptomyces purpurascens, extracting the culture filtrate and mycelium in a neutral medium and obtaining the antibiotic from the extracts by known methods. The antibiotic is found to possess excellent activity against a broad class of organisms, especially mycoplasmas and gram-positive bacteria.

United States Patent 1 Brockmann et al.

[54] BETA-RHODOMYCIN V [75] Inventors: Hans Brockmann, Goettingen;Martin Scheer, Wuppertal-Elberfeld, both of Germany [73] Assignee:Farbenfabriken Bayer Aktiengesellschait, Leverkusen, Germany 221 Filed:Mar. 12, 1971 21 Appl.No.: 123,882

[30] Foreign Application Priority Data Mar. 18, 1970 Germany ..P 20 12808.9

[52] U.S. Cl. ..260/210 AB, 424/180,195/80 [51] Int. Cl ..C07c 47/18[58] Field of Search ..260/210 AB, 210 R [56] References Cited OTHERPUBLICATIONS Bayer, Chem. Abst., Vol.53, 1959, p. 4663(b). Brockmann etal., Chem. Abst., Vol. 70, 1969, p. 97ll6y.

[ 3,723All 51 Mar. 27, 1973 Primary ExaminerLewis Gotte AssistantExaminer-Johnnie R. Brown AttorneyMcCarthy, DePaoli, OBrien & Price [57]ABSTRACT An antibiotic designated B-rhodomycin V and corresponding tothe formula O-rliodosuminc OR OH O OH wherein R represents atetraglycoside consisting of 2 mols rhodinose, 1 mol 2-desoxy-fucose and1 mol rhodosamine is prepared by cultivating Streptomyces- 1 Claim, NoDrawings BETA-RHODOMYCIN v BACKGROUND INVENTION 1 Field of the InventionThe present invention relates in general to the field of antibiotics,more particularly to a new member of the class of rhodomycins.

2. Description of the Prior Art The rhodomycins are closely interrelatedand are nitrogen-containing bases which usually occur as mixtures. Theyare red dyestuffs.

German Patent No. 851,398 is directed to a process for the production ofantibiotically active substances, which is characterized in thatstreptomycetes, especially Streptomyces purpurascens novv spec., areallowed to grow under aerobic conditions on or in nutrient media knownfor the cultivation of streptomycetes and the rhodomycins are obtainedfrom the resultant cultures by usual methods.

The subject matter of German Patent No. 913,813 is the preparation of acrystallized rhodomycin hydrochloride, designated therein asrhodomycin-A hydrochloride.

SUMMARY OF THE INVENTION The subject-matter of the present inventionis anew B-rhodomycin called B-rhodomycin V and correspondin to the formula Rtetraglycoside from 2 mol rhodinose, 1 mol 2-desoxy-fucose and 1 molrhodosamine.

This new rhodomycin is distinguished from the known rhodomycins in thatit contains fivesugar radicals, whereas the known rhodomycins, nowcalled B- rhodomycins I and B-rhodomycins II, only contain one and twosugar radicals, respectively [Tetrahedron Letters, 1969, pages 415 to419].

According to the invention, the new rhodomycin is prepared bycultivating Streptomyces purpurascens, extracting the culture filtrateand vthe mycelium in a neutral medium, and obtaining B-rhodomycin V fromthe extracts in known manner.

The process according to the invention is thus distinguished from theknown processes for the production of the known rhodomycins in that theextraction is here carried out in a neutral medium whereas in theearlier patents mentioned above the extraction is carried out in anacidic medium.

All species of streptomycetes are suitable starting materials for theprocess according to the invention. Streptomyces purpurascens Ist 313 isparticularly suitable. Neutral extraction means in the present case anextraction at a pH value of 6.5 to 7.5.

fi-Rhodomycin V has an excellent chemotherapeutic, especiallyantibacterial activity, as can be seen from the following results oftests in vitro.

Testing of the minimal inhibition concentration was carried out for thegram-positive and gram-negative causative organisms in the KLEIN medium(meat extract, peptone, dextrose, pH 7.1) at an incubation temperatureof 37C. for 24 hours, number of seeded germs l0 genns/ml; and formycoplasmas in PPLO broth (PPLO basic substrate, dextrose, equine serum,pl-l7.6) at an incubation temperature of 37C. for 48 hours to 72 hours,number of seeded germs 10 germs/ml.

TABLE 1 Minimal Inhibition Concentration (MIC) (y/ml nutrient medium) of,B-rhodomycin V of Type of germ strains MIC -y/ml Streptococci ofdifferent species 5 0.015 to 1.00 Staphylococcus aureus 4 0.050 to 0.12Sarcinae 2 0.060 to 1.00 Corynebacterium pyogener 2 0.060 to 0.12Pseudomona: aemginosa 2 E. coli 2 50 Klebsiella pneumom'ae l 25Salmonella spec. 2 50 to 200 Parteurella multocida 2 6.25

Bordetella bronchiseptica 1 50 Alkaligenenesfaecalilr 1 25 Mycoplasmasavian mycoplasmas 4 0.001 to 0.002 bovine mycoplasmas 2 0.030 to 0.060porcine mycoplasmas 2 0.001 to 0.002

The values of the Table show that B-rhodomycin V has an excellent andunusually wide-ranging activity, especially against mycoplasmas andgram-positive bacteria.

The excellent and wide-ranging activity permits of their use in human aswell as veterinary medicine; they can be used for the prophylaxis ofbacterial infections but also for the treatment of already existingbacterial infections.

As indications for the field of veterinary medicine, there may bementioned, for example, infections with mycoplasmas and intestinalinfections. For prophylaxis, the B-rhodomycin V can be used as admixtureto feeding stuff.

EXAMPLE 1 A 10 liters preliminary culture of Streptomyces purpurascenslst 313 is incubated on a nutrient solution with 2 percent of soya flourand 2 percent mannitol submersed at 26C. for 24 hours, transferred to350 liters of a nutrient solution of the same composition, and fermentedfor 96 hours at 260C. with stirring and aeration.

When the fermentation has been terminated, the redbrown culture broth isstirred with 20 kg kieselguhr and filtered. The culture filtrate and themycelium are worked up separately. The mycelium is stirred three timeswith 60 liters of acetone each time, the combined orange-red acetoneextracts are evaporated in a vacuum at 50C. to a volume of 30 liters,extracted three times with 15 liters of butanol each time, and thecombined butanol extracts are concentrated in a vacuum at 50C. to volumeof about 3 liters. The remaining red-brown oil is taken up with 10liters chloroform (solution A).

The culture filtrate (350 liters, pH 7.0 to 7.5) is extracied with 150liters butanol and the deep-red butanol phase is concentrated in avacuum at 50C. until there remain about 3 liters of a deep-red oil whichis taken up with 10 liters chloroform (solution B).

The combined chloroform solutions A and B are distributed over six 100 X10 cm columns of carboxymethyl cellulose (deposited with 2N hydrochloricacid; washed out (1) with water until the reaction is neutral, (2) withacetone and (3) with chloroform). Rinsing with 5 to liters chloroformper column brings fats, anthracyclinones and non-basic anthracyclinesinto the filtrate.

The portion remaining on the column is eluted with 10 percent aqueouspotassium hydrogen phosphate, and the deep-red eluate (5 liters percolumn) which has been adjusted to pH 8.0 with dilute ammonia isextracted three times with three liters of chloroform each time. Thecombined chloroform phases are evaporates to dryness in a vacuum, theresidue is taken up with a little chloroform, and the red B-rhodomycin Vis precipitated with petroleum ether (40 to 50C.). Yield 150 to 170 mgper liter of culture broth.

2 g of the B-rhodomycin V preparation obtained as described above arechromatographed through a 100 X 4 cm cellulose column in a systembutanol-0.7 m phosphate buffer pH 5.8 1:1 The main zone (migratingfastest) is brought into the filtrate by rinsing with r phase, diluteammonia is then added to the filt; zte until the pH is 8.0, the mixtureis shaken with chloroform, and the chloroform phase, dried over sodiumsulphate, is evaporated to dryness in a vacuum. The

B-rhodomycin V remaining behind as a red lacquer is taken up with alittle chloroform and precipitated in the form of a red powder or alsoof red needles at boiling temperature with cyclohexane. Melting point173 to 175C. (decomposition); [a 1 m 40 i 2C, 0.2 in chloroform. C H N O1059.2)

Calc.: C 61.14; H 7.36; N 2.61

found: C 61.30; H 7.40; N 2.65 dried in a high vacuum at C. for 15hours.

IR (KBr compact) 3450, 2910, 1730, 1595/cm.

The B-rhodomycin V is readily soluble in methanol, butanol, acetone,chloroform, 0.7 m phosphate buffer pH 5.8, dilute acids; it is sparinglysoluble in benzene, very sparingly soluble in water and virtuallyinsoluble in cyclohexane.

What is claimed is:

1. B-Rhodomycin V of the formula on o CHJCI'IQ I 1 My 1 on on 3 on inwhich R stands for a tetraglycoside radical consisting of 2 molrhodinose, 1 mol 2-desoxy-fucose and 1 mol rhodosamine.

